Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous inherited degenerative retinopathies caused by abnormalities of photoreceptors or retinal pigment epithelium in the retina leading to progressive sight loss. Rhodopsin is the prototypical G-protein-coupled receptor located in the vertebrate retina and is responsible for dim light vision. Here, novel M39R and N55K variants were identified as causing an intriguing sector phenotype of RP in affected patients, with selective degeneration in the inferior retina. To gain insights into the molecular aspects associated with this sector RP phenotype, whose molecular mechanism remains elusive, the mutations were constructed by site-directed mutagenesis, expressed in heterologous systems, and studied by biochemical, spectroscopic, and functional assays. M39R and N55K opsins had variable degrees of chromophore regeneration when compared with WT opsin but showed no gross structural misfolding or altered trafficking. M39R showed a faster rate for transducin activation than WT rhodopsin with a faster metarhodopsinII decay, whereas N55K presented a reduced activation rate and an altered photobleaching pattern. N55K also showed an altered retinal release from the opsin binding pocket upon light exposure, affecting its optimal functional response. Our data suggest that these sector RP mutations cause different protein phenotypes that may be related to their different clinical progression. Overall, these findings illuminate the molecular mechanisms of sector RP associated with rhodopsin mutations.

The three cloned galanin receptors show a higher affinity for galanin than for galanin N-terminal fragments. Galanin fragment (1-15) binding sites were discovered in the rat Central Nervous System, especially in dorsal hippocampus, indicating a relevant role of galanin fragments in central galanin communication. The hypothesis was introduced that these N-terminal galanin fragment preferring sites are formed through the formation of GalR1-GalR2 heteromers which may play a significant role in mediating galanin fragment (1-15) signaling. In HEK293T cells evidence for the existence of GalR1-GalR2 heteroreceptor complexes were obtained with proximity ligation and BRET2 assays. PLA positive blobs representing GalR1-GalR2 heteroreceptor complexes were also observed in the raphe-hippocampal system. In CRE luciferase reporter gene assays, galanin (1-15) was more potent than galanin (1-29) in inhibiting the forskolin-induced increase of luciferase activity in GalR1-GalR2 transfected cells. The inhibition of CREB by 50 nM of galanin (1-15) and of galanin (1-29) was fully counteracted by the non-selective galanin antagonist M35 and the selective GalR2 antagonist M871. These results suggested that the orthosteric agonist binding site of GalR1 protomer may have an increased affinity for the galanin (115) vs galanin (1-29) which can lead to its demonstrated increase in potency to inhibit CREB vs galanin (1-29). In contrast, in NFAT reporter gene assays galanin (1-29) shows a higher efficacy than galanin (115) in increasing Gq/11 mediated signaling over the GalR2 of these heteroreceptor complexes. This disbalance in the signaling of the GalR1-GalR2 heteroreceptor complexes induced by galanin (1-15) may contribute to depression-like actions since GalR1 agonists produce such effects. (C) 2014 Elsevier Inc. All rights reserved.

Mercuric compounds were previously shown to affect the visual phototransduction cascade, and this could result in vision impairment. We have analyzed the effect of mercuric chloride on the structure and stability of the dim light vision photoreceptor rhodopsin. For this purpose, we have used both native rhodopsin imrnunopurified from bovine retinas and a recombinant mutant rhodopsin carrying several Cys to Ser substitutions in order to investigate the potential binding site of mercury on the receptor. Our results show that mercuric chloride dramatically reduces the stability of dark-state rhodopsin and alters the molecular features of the photoactived conformation obtained upon illumination by eliciting the formation of an altered photointermediate. The thermal bleaching kinetics of native and mutant rhodopsin is markedly accelerated by mercury in a concentration-dependent manner, and its chromophore regeneration ability is severely reduced without significantly affecting its G-protein activation capacity. Furthermore, fluorescence spectroscopic measurements on the retinal release process, ensuing illumination, suggest that mercury impairs complete retinal release from the receptor binding pocket. Our results provide further support for the capacity of mercury as a hazardous metal ion with reported deleterious effect on vision and provide a molecular explanation for such an effect at the rhodopsin photoreceptor level. We suggest that mercury could alter vision by acting in a specific manner on the molecular components of the retinoid cycle, particularly by modifying the ability of the visual photoreceptor protein rhodopsin to be regenerated and to be normally photoactivated by light.

The molecular chaperone Hsp90 is important for the functional maturation of many client proteins, and inhibitors are in clinical trials for multiple indications in cancer. Hsp90 inhibition activates the heat shock response and can improve viability in a cell model of the P23H misfolding mutation in rhodopsin that causes autosomal dominant retinitis pigmentosa (adRP). Here, we show that a single low dose of the Hsp90 inhibitor HSP990 enhanced visual function and delayed photoreceptor degeneration in a P23H transgenic rat model. This was associated with the induction of heat shock protein expression and reduced rhodopsin aggregation. We then investigated the effect of Hsp90 inhibition on a different type of rod opsin mutant, R135L, which is hyperphosphorylated, binds arrestin and disrupts vesicular traffic. Hsp90 inhibition with 17-AAG reduced the intracellular accumulation of R135L and abolished arrestin binding in cells. Hsf-1(/) cells revealed that the effect of 17-AAG on P23H aggregation was dependent on HSF-1, whereas the effect on R135L was HSF-1 independent. Instead, the effect on R135L was mediated by a requirement of Hsp90 for rhodopsin kinase (GRK1) maturation and function. Importantly, Hsp90 inhibition restored R135L rod opsin localization to wild-type (WT) phenotype in vivo in rat retina. Prolonged Hsp90 inhibition with HSP990 in vivo led to a posttranslational reduction in GRK1 and phosphodiesterase (PDE6) protein levels, identifying them as Hsp90 clients. These data suggest that Hsp90 represents a potential therapeutic target for different types of rhodopsin adRP through distinct mechanisms, but also indicate that sustained Hsp90 inhibition might adversely affect visual function.

11-cis-retinal acts as an inverse agonist stabilizing the inactive conformation of visual pigments, and upon photoactivation, it isomerizes to all-trans-retinal, initiating signal transduction. We have analyzed opsin regeneration with retinal analogs for rhodopsin and red cone opsin. We find differential binding of the analogs to the receptors after photobleaching and a dependence of the binding kinetics on the oligomerization state of the protein. The results outline the sensitivity of retinal entry to the binding pocket of visual receptors to the specific conformation adopted by the receptor and by the molecular architecture defined by specific amino acids in the binding pocket and the retinal entry site, as well as the topology of the retinal analog. Overall, our findings highlight the specificity of the ligand-opsin interactions, a feature that can be shared by other G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) are a widespread family of transmembrane receptors with different physiologically relevant functions. Alterations in the structure and function of these receptors at different levels (ligand binding, signaling and trafficking) may result in a number of pathological conditions which represent a major health problem. Mutations in these receptors are also linked to different inherited diseases for which there is no cure to date. Rationale design, based on receptor structural knowledge, is needed for the discovery of novel drugs with higher selectivity and less side effects. In fact, about 50% of the drugs currently under development target this kind of receptors. Oligomerization among GPCRs has been clearly established from experimental, particularly in vitro, studies. Moreover, homo and heterodimerization provide new unexpected clues for explaining the molecular mechanisms underlying some diseases in which GPCRs signaling might be affected. In this review we will analyze GPCRs structure and function for a better understanding of the dimerization process and the experimental approaches currently used to detect such interactions. Furthermore, how drugs targeting heteromers can represent new opportunities to tackle novel and safer treatments of some pathologies will be described. Recent results, in this regard, will be reported as encouraging examples in the field. Finally, the newest technologies available for developing drugs targeting heteromers will also be reviewed highlighting the importance of bivalent ligands that emerge as very powerful molecules interacting with heteromers.

Galanin receptor 1 (GalR1) is a G-protein coupled receptor (GPCR) widely distributed in the brain which elicits a wide range of biological effects via interactions with its cognate ligand galanin. Galanin is a 30-mer neuropeptide with an N-terminal region comprising about 15 highly conserved residues, which acts as the crucial region for agonist-receptor binding. Moreover, it has been recently shown that GalR1 could be involved in other brain processes due to its interactions with other GPCR via heterodimerization. The 5-HT1A serotonin receptor heterodimerizes with GalR1 receptor, and this interaction may modulate the molecular mechanism of depression in which this serotonin receptor is involved.
We have explored how galanin ligand binding affects GalR1 heterodimerization with 5-HT1A, specially the difference between the native peptide and the N(1-15) terminal peptide, by means of FRET spectroscopy in HEK293 transiently transfected cells. We have also used synthetic peptides with sequential L-Ala substitutions of individual amino acids in the human galanin sequence and examined their effect on heterodimerization, especially for amino acids at positions (1-4) and (9-14) which seem to be essential for binding and functional coupling of the receptor. By using a construct where the 1D4 antigenic region has been fused to GalR1 using tailed PCR, we have been able to immunopurify this receptor from HEK293 cells, in order to analyze structural changes induced by the binding of the different galanin L-Ala variants using fluorescence spectroscopy. Finally, we have also probed GalR1-GalR2 heterodimerization by means of FRET.

Rhodopsin and cone opsins, the visual pigments of the vertebrate retina are a distinct group of G-protein-coupled receptors which are covalently bound to their ligand, the chromophoric 11-cis-retinal in the resting inactive state. The retinal binding site of these receptors is very dynamic allowing their conformational stabilization and functional activation. Though various retinal analogs were reported to behave as chromophores for retinal receptors, their specific physical properties and activation ability varies among them. We have carried out a study of the dynamics of the retinal binding site of rhodopsin and cone opsins after photo-activation which had not been previously analyzed. In order to do that, the photoreceptor genes inserted into the pMT4 vector were transiently transfected in COS-1 cells. Opsins regenerated with 11-cis-retinal were immunopurified by using rho-1D4 antibody. Fluorescence spectroscopy was employed to monitor conformational changes upon ligand binding to the receptor with retinal analogs 11-cis-retinal and 9-cis-retinal and their functional behavior was measured using a G-protein activation assay. We found that, after photo-activation, both retinals can access the binding pocket of red cone opsin but only 9-cis-retinal can occupy the retinal binding site of rhodopsin. The binding kinetics of 9-cis-retinal to photo-activated rhodopsin showed a faster entry into the retinal binding site followed by a binding process with t1/2 of ~1 min. Furthermore, the G-protein activation profile of post-bleached red cone and rhodopsin reveal differences which may account for the different rod and cone sensitivities. In summary, the present study suggests that the rhodopsin retinal binding site is less conformationally flexible than the red cone binding pocket which may be more open to permit the entry of both retinal analogs.

Rhodopsin is the photoreceptor located in the rod cells of the retina. It has seven transmembrane helices and is a prototypic member of the G protein-coupled receptor superfamily. The structures and functions of these receptors are clearly affected by the lipid composition of the cell membrane, and their study in a purified recombinant form is usually performed in detergent solution. There is a need to study these receptors in a physiologically relevant environment because the lipid environment is known to have an important effect on their function. In this work, rhodopsin reconstituted in docosahexaenoic acid (DHA) liposomes is shown to have more thermal stability than when it is solubilised with the neutral detergent dodecyl maltoside. Moreover, the specific interaction between rhodopsin and DHA was followed by means of Langmuir experiments with insertion of rhodopsin into lipid monolayers; this showed high affinity for the lipid-receptor interaction...

G protein-coupled receptors (GPCRs) play critical roles in cellular processes and signaling and have been shown to form heteromers with diverge biochemical and/or pharmacological activities that are different from those of the corresponding monomers or homomers. However, despite extensive experimental results supporting the formation of GPCR heteromers in heterologous systems, the existence of such receptor heterocomplexes in the brain remains largely unknown, mostly because of the lack of appropriate methodology. Herein, we describe the in situ proximity ligation assay procedure underlining its high selectivity and sensitivity to image GPCR heteromers with confocal microscopy in brain sections. We describe here how the assay is performed and discuss advantages and disadvantages of this method compared with other available techniques.

Rhodopsin and cone opsins, the photosensitive pigments of the vertebrate retina are a
distinct group of G-protein-coupled receptors which are covalently bound to a
chromophore, 11-cis-retinal. These receptors play a central role in the visual
phototransduction process mediating dim-light vision and colour vision respectively.
The kinetics of the response of the rod photoreceptor rhodopsin and its related cone
opsins is very different suggesting important differences in both inter and
intramolecular interactions. Photoactivation of these receptors leads to G-protein
binding and activation and eventual retinal release from its binding pocket which
causes structural rearrangements on the opsin apoproteins. Although the effect of
various retinal analogs were reported to mimic -at least partially- the action of 11-cisretinal
with opsins, the effect of retinal analogs on rhodopsin and cone opsins after
photoactivation -at the post-bleaching level- has not been characterized in detail.
This process is important for the regeneration of the fully-functional receptors after
chromophore binding, and it is central to the desensitization mechanism of the
photoreceptor cells. Rhodopsin and cone opsin genes -cloned into pMT4 plasmidwere
transiently transfected and expressed in eukaryotic COS-1cells. Opsins were
regenerated with 11-cis-retinal and purified by immunoaffinity chromatography using
the Rho-1D4 monoclonal antibody coupled to a Sepharose matrix in dodecyl maltoside
detergent solution. The rates of receptor reconstitution with 11-cis-retinal, and the
retinal analog 9-cis-retinal, with rhodopsin and red cone opsin respectively, after
bleaching, were analyzed by means of fluorescence spectroscopy.
The results showed that, after bleaching, 9-cis-retinal can access the binding pocket of
rhodopsin but 11-cis-retinal cannot. In contrast, both retinals can access the red cone
opsin binding pocket of the photobleached receptor even long time after bleaching.
We conclude that post-bleaching retinal reconstitution of opsins (rhodopsin and red
cone opsin) reflects a variable degree of conformational flexibility that allows to
preferentially accommodate certain retinal analogs. Studies are underway to
incorporate these receptors into liposomes and to determine their ability to activate
their corresponding G-proteins.

The visual photoreceptor rhodopsin undergoes a series of conformational changes upon light activation, eventually leading to the active metarhodopsin II conformation, which is able to bind and activate the G-protein,
transducin. We have previously shown that mutant rhodopsins G51V and G89D, associated with retinitis pigmentosa, present photobleaching patterns characterized by the formation of altered photointermediates whose
nature remained obscure. Our current detailed UV–visible spectroscopic analysis, together with functional characterization, indicate that these mutations influence the relative stability of the different metarhodopsin photointermediates by altering their equilibria and maintaining the receptor
in a nonfunctional light-induced conformation that may be toxic to photoreceptor cells. We propose that G51V and G89D shift the equilibrium from
metarhodopsin I towards an intermediate, recently named as metarhodopsin Ib, proposed to interact with transducin without activating it. This may be one of the causes contributing to the molecular mechanisms underlying cell death associated with some retinitis pigmentosa mutations.

Rhodopsin is the visual photoreceptor responsible for dim light vision. This receptor is located in the rod cell of the retina and is a prototypical member of the G-protein-coupled receptor superfamily. The structural details underlying the molecular recognition event in transducin activation by photoactivated rhodopsin are of key interest to unravel the molecular mechanism of signal transduction in the retina. We
constructed and expressed rhodopsin mutants in the second and third cytoplasmic domains of rhodopsin –where the natural amino acids were substituted by the human M3 acetylcholine muscarinic receptor homologous residues– in order to determine their potential involvement in G-protein recognition. These mutants showed normal chromophore formation and a similar photobleaching behavior than WT rhodopsin,
but decreased thermal stability in the dark state. The single mutant V1383.53 and the multiple mutant containing V2275.62 and a combination of mutations at the cytoplasmic end of transmembrane helix 6 caused a reduction in transducin activation upon rhodopsin photoactivation. Furthermore, combination of mutants at the second and third cytoplasmic domains revealed a cooperative role, and partially restored transducin activation. The results indicate that hydrophobic interactions by V1383.53,
V2275.62, V2506.33, V2546.37 and I2556.38 are critical for receptor activation and/or efficient rhodopsin–transducin interaction.